1. Root Hairless 1 (RHL1)
An RHL1 polypeptide was first described in 1998 by Schneider at al. (Genes Dev. 12, 2013-2021) as a nuclear targeted protein required for root hair initiation in Arabidopsis thaliana. RHL1 polypeptides are ubiquitous to the viridiplantae kingdom. Sequence comparison of RHL1 originating from different organism reveals that RHL1 polypeptides share an overall sequence similarity around 30-80% identity. RHL1 polypeptides comprise a number of putative nuclear localization signals as well as phosphorylation sites and a PEST sequence which is a putative proteasome-dependent proteins degradation motif. The presence of such motifs may reportedly confer some regulatory roles by modulating subcellular localization of topos and for their interaction with other proteins. The C-terminus of RHL1 proteins has weak but significant sequence similarity to the C-terminal of mammalian Topo II-alpha protein (Sugimoto-Shirasu et al. 2005 PNAS 102, 18736-17741). Eukaryotic topo II proteins belong to the subclass of the type II topo (typeIIA) that is required to unwind replicating double-stranded DNA. Physical Interaction between an RHL1 polypeptide and a plant topo VI protein, At TOP6B, has been reported (Sugimoto-Shirasu et al. 2005). It has been suggested that RHL1 polypeptides function in a plant topo VI complex active during the mitotic cell cycle and endocycle of plant cells. Arabidopsis thaliana plants, hyp7, carrying mutations in an RHL1 gene exhibit an extreme drawf phenotype and defects in endoreduplication (Sugimoto-Shirasu et al. 2005).
2. Transglutaminases (TGases)
Transglutaminases (TGases, EC 2.3.2.13; protein-glutamine-gamma-glutamyltransferase) are a family of enzymes that have a range of calcium (Ca)-dependent catalytic activities, most of which concern the post-translational modification of proteins. They catalyze the covalent attachment to proteins and polypeptides of a series of substances containing primary amine groups, i.e., they promote the formation of amide linkages, generally in a Ca-dependent fashion, between the primary amine of an amine donor substrate and the y-carboxamide group of peptide-bound
endo-glutamine residues in proteins or polypeptides that are the amine acceptors:protein glutamine+alkylamine=protein N5-alkylglutamine+NH3.
Polyamines have been shown to serve as physiological substrates of TGases. Polyamines appear to play an essential role in growth and cell division process in animals, microorganisms, and plants. One of the roles of polyamines is their regulatory action by a TGase-mediated process of post-translational modification (addition of polyamine moieties) of enzymes and structural proteins.
TGases enzymes are found intracellularly and extracellularly, and are widely distributed in bacteria, animals and plants. In plants, the TGase activity is found in chloroplasts. Rubisco and apoproteins of the antenna complex have been shown to be substrates of TGase activity, thereby suggesting a role of these enzymes in photosynthesis related processes, such as protection of photosystem antenna proteins (Villalobos et al. (2004) Gene 336: 93-104).
Transgenic rice plants (Claparols et al. (2004) Transgenic Research 13: 195-199) expressing a gene encoding rat prostate calcium-dependent transglutaminase polypeptide under the control of maize constitutive promoter accumulated the recombinant enzyme in an inactive form.
International patent application WO 2003/102128 describes a nucleic acid sequence encoding a corn TGase polypeptide, vectors, micro-organisms and plants comprising such nucleic acid sequences, and the use of polypeptides with such TGase activity in food manipulation, processing and transformation.
Surprisingly, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a TGase polypeptide as defined herein, gives plants having increased seed yield-related traits relative to control plants.
According to one embodiment, there is provided a method for increasing seed yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a TGase polypeptide as defined herein. The increased seed yield-related traits comprise one or more of: increased total seed yield per plant, increased number of filled seeds, and increased harvest index.
3. Tryptichon (TRY-Like)
The Arabidopsis gene Tryptichon encodes a protein that reportedly negatively regulates trychome development and positively regulates root hair development. Trichome patterning in Arabidopsis is a model for the generation of a spacing pattern from initially equivalent cells. Schellmann et al. (EMBO J. 21, 5036-5046, 2002) show that the Tryptichon gene that functions in lateral inhibition encodes a single-repeat MYB-related transcription factor that lacks a recognizable activation domain. It has high sequence similarity to the root hair patterning gene Caprice. Both genes are expressed in trichomes and act together during lateral inhibition. They further show that Tryptichon and Caprice act redundantly in the position-dependent cell fate determination in the root epidermis. Thus, the same lateral inhibition mechanism seems to be involved in both de novo patterning and position-dependent cell determination (Schellmann et al., 2002).
4. Brassinazole Resistant1 (BZR1)
The regulation of gene expression is key to the viability of any cell. Several hundreds of proteins are involved in the regulation of gene transcription. In particular transcription factors play a central role and act directly on gene promoters. Plant genomes devote approximately 7% of their coding sequence to transcription factors (TFs; Rushton et al. 2008 Plant Physiology 147:280-295 (2008).
Plants encode a particular class of transcription factors, the BES or BZR proteins, which modulate gene response to fluctuations in plant steroid hormones such as brassinosteroids (BRs). BZR transcription factors (BZR TFs) are characterized by the presence of a conserved BZR1 repressor domain typically found at the N-terminus of the protein and involved in binding to the targeted gene promoter. Plant typically encode a small number of BZR TFs. For example the Arabidopsis genome contains only 6 genes encoding BZR TFs, while tobacco, a plant in which this family of TFs is expanded encodes 19 BZR TFs. All TFs comprised a conserved BZR1 repressor domain and are predicted to function in the modulation of BR signalling.
In Arabidopsis thaliana, the cascade of events in BR signalling are triggered upon binding of BRs to the BRASSINOSTEROID INSENSITIVE1 (BRI1)/BKI1 receptor complex at the plasma membrane, causing the release of BKI1. The subsequent dimerization of BRI1 and BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) activates a downstream signal transductionpathway that leads to BRI1 EMS SUPPRESSOR1 (BES1) and BRASSINAZOLE RESISTANT1 (BZR1). The phosphorylation of BES1 and BZR1 by the kinase BIN2 appears to control their signalling activity by acting on the subcelullar localization and stability of the protein. Dephosphorilated BZR1 accumulates in the nuclei which is the site at which the transcriptional function is performed (Wang et al., 2006 Cell Res. 16: 427-434). Mechanistically, transcription factors of the BZR1 family directly bind to the promoter of the targeted gene and may act to activate or repress expression.
Methods for modulating the Brassinosteroid response pathway to modify a number of traits in plants have been disclosed (U.S. Pat. No. 6,921,848). The traits as defined in U.S. Pat. No. 6,921,848 comprised increased growth and cell elongation in various organs and tissues. However those effects did not result in an increase in the number of organs such as the number of seeds produced and/or in an increase in the seed yield of the plant.